This invention relates in general to a modified serum albumin useful as a blood volume expander, and in particular to a recombinant or otherwise modified serum albumin that is mutated such as by truncation, elongation or insertion at its n-terminal region which has improvements in its metal binding properties and reduced affinity for metal elements such as nickel or copper and which can thus be used safely and effectively in a variety of applications including use as a blood volume expander, for excipient and culture media applications, or as additives or substitutes in a wide range of products including medicines, cosmetics, dairy products and dietary supplements which would normally utilize unmodified forms of albumin or related compounds.
The serum albumins belong to a multigene family of proteins that includes alpha-fetoprotein and human group-specific component, also known as vitamin-D binding protein. The members of this multigene family are typically comprised of relatively large multi-domain proteins, and the serum albumins are the major soluble proteins of the circulatory system and contribute to many vital physiological processes. Serum albumin generally comprises about 50% of the total blood component by dry weight, and as such is responsible for roughly 80% of the maintenance of colloid osmotic blood pressure and is chiefly responsible for controlling the physiological pH of blood.
The albumins and their related blood proteins also play an extremely important role in the transport, distribution and metabolism of many endogenous and exogenous ligands in the human body, including a variety of chemically diverse molecules including fatty acids, amino acids, steroids, calcium, metals such as copper and zinc, and various pharmaceutical agents. The albumin family of molecules are generally thought to facilitate transfer many of these ligands across organ-circulatory interfaces such as the liver, intestines, kidneys and the brain, and studies have suggested the existence of an albumin cell surface receptor. See, e.g., Schnitzer et al., P.N.A.S. 85:6773 (1988). The albumins are thus intimately involved in a wide range of circulatory and metabolic functions.
Human serum albumin (HSA) is a protein of about 66,500 kD and is comprised of 585 amino acids including at least 17 disulphide bridges. As with many of the members of the albumin family, human serum albumin plays an extremely important role in human physiology and is located in virtually every human tissue and bodily secretion. As indicated above, HSA has an outstanding ability to bind and transport a wide spectrum of ligands throughout the circulatory system including the long-chain fatty acids which are otherwise insoluble in circulating plasma. The atomic structure and particular details regarding the binding affinities of albumin and the specific regions primarily responsible for those binding properties have been previously determined as set forth, e.g., in U.S. patent application Ser. No. 08/448,196, filed May 25, 1993, now U.S. Pat. No. 5,780,594 and U.S. patent application Ser. No. 08/984,176, filed Dec. 3, 1997, now U.S. Pat. No. 5,948,609, both of which are incorporated herein by reference.
In addition to human serum albumin, studies have been made on albumins in a variety of animal species, and it has been determined that over 60% of the amino acid sequences are conserved among the known albumin sequences of many mammals such as bovine, rat and human serum albumin. Moreover, as more and more albumins from other animal species have been sequenced, it has been found that the albumins from a wide range of vertebrate species including sheep, frogs, salmon, mice, pigs and even sea lampreys share varying degrees of sequence homology, and all share the characteristic repeating pattern of disulphide bridges observed in human serum albumin, thus implying a common three-dimensional structure. Furthermore, all members of the albumin multigene family for which sequences have been determined appear to have internal sequence homology (from two- to seven-fold), thus suggesting that the proteins evolved from a common ancestral protein, and reflecting the vital nature and function of this protein. See, e.g., Carter et al., Science 244:1195 (1989).
Because of the vital role played by albumins, there are literally thousands of applications for serum albumin and its related proteins covering a wide range of physiological conditions, and most often, native serum albumin has been used. However, unlike blood proteins such as hemoglobin, native serum albumins are non-functional as oxygen transport systems, and thus have not been useful in blood replacement systems requiring oxygen transport. More recently, an oxygen-transporting albumin-based blood replacement composition was developed which can be utilized as a blood volume expander, as has been disclosed in U.S. Pat. No. 5,948,609, incorporated herein by reference, and this composition further increases the importance and usefulness of serum albumin.
Additionally, in applications involving albumin, it has been known to utilize the human serum albumin sequence of the prototypical or major allotype of the human serum albumin sequence, which has included the well known n-terminal amino add sequence n-DAHK-c. See, e.g., Carter et al., Advances in Protein Chemistry, Vol. 45, 153-203 (1994) and Peters, xe2x80x9cAll About Albuminxe2x80x9d, Academic Press (1995). The binding of copper and nickel to the n-terminal peptide of albumins has been known and studied for many years, and this site has been designated as functional binding location Site VI in Carter et al. (1994). The sequence X-X-Histidine appears to be the key to the copper and nickel metal binding at this site, and of additional importance is the structural flexibility of the n-terminal polypeptide which cannot be structurally hindered.
At physiological pH, copper is bound with extraordinarily high affinity to this site (Ka=1.6xc3x971016 Mxe2x88x921) (see Camerman et al., Can. J. Chem., Vol. 54:1309-1316 (1976)), perhaps the highest reported binding constant of any bound ligand to serum albumin. By comparison, the binding for Nickel is Ka=3xc3x97105 Mxe2x88x921. See Lau et al., J. Biol. Chem. Vol. 249:5878-5884 (1974). While this feature of the albumin molecule serves to protect the body from the potential damaging influences of the metals, especially copper, the nickel complex with albumin is known to elicit allergic reactions in some individuals, which occurs following ingestion of Ni(II) or exposure to nickel plated jewelry or other similar items. For example, an occupational asthma resulting from nickel binding is well recognized and has been traced to antibodies against Ni(II) specifically bound to the n-terminus of human serum albumin. See Carter et al. (1994), supra and Nieboer et al., Br. J. Ind. Med., Vol. 41:56-63 (1984).
In the normal course of recombinant production of albumin and other proteins, there is usually a given level of certain metals, including nickel and copper, which are required as components of the culture media and used in albumin production. Consequently, a significant level of nickel, copper and/or other metals is chelated by the n-terminal peptide of albumin during production, as evidenced during production by the green and yellow coloration of the recombinant human serum albumin. However, the presence of these metals, even in trace amounts, in the albumin produced via recombinant methods can lead to significant health problems as indicated above.
There is thus a significant need to develop safe and effective serum albumin products for use in many applications, particularly those which involve use in humans either internally or externally, which can reduce or eliminate the high affinity of albumin to copper and nickel and/or other metals, and which can thus reduce the risk that a potential albumin-based product will elicit an allergic response to the bound metal in a human or animal who is being treated with an albumin composition.
Accordingly, it is thus an object of the present invention to provide a novel serum albumin composition in which the affinity to trace metals such as nickel and/or copper is reduced or eliminated.
It is further an object of the present invention to provide a novel modified serum albumin having reduced affinity to trace metals which can be utilized in a variety of applications including as a blood replacement product, a cosmetic, a medicament, or a pharmaceutical additive.
It is still further an object of the present invention to provide a novel serum albumin product which has improved clarity so as to become more useful in scientific applications involving spectroscopy.
It is even further an object of the present invention to provide a novel serum albumin-based blood replacement product which not only encompasses a variety of useful physiological functions including oxygen transport and expansion of blood volume, but which can be utilized safely in humans and animals and which has reduced or eliminated binding to trace metals such as nickel and/or copper and thus presents a greatly reduced risk of causing an allergic reaction or other harmful conditions.
These and other objects are achieved by virtue of the present invention which provides a modified serum albumin which has been either truncated by at least one amino acid at its n-terminal end or which has been mutated in such a way as to disrupt the metal binding site of the serum albumin previously identified as binding site VI. These changes and mutations to this binding site include elongation, insertion or other changes to the n-terminal end, such as to the histidine at amino acid position 3, which are sufficient so as to either sterically hinder the binding site V or eliminate vital binding interactions, and thus reduce the affinity of this region to metals such as nickel and/or copper. As a result of these modifications, which may be made either recombinantly or through other suitable physical or chemical means, the resulting serum albumin composition will have greatly reduced or totally eliminated binding to the trace metals, and can thus be used safely and effectively in a variety of applications, including as a blood product, a cosmetic, a medicament, a pharmaceutical additive, or numerous other applications which presently employ albumin compositions.
These and other features of the present invention are set forth in, or will become obvious from, the detailed description of the preferred embodiments provided hereinbelow.